Nanoparticles for administering an active ingredient

Abstract

Nanoparticles (1) for administering at least one active ingredient (4), said nanoparticles comprising a magnetizable core (2), the core (2) having a shell (3) made of a polyphosphate and said shell (3) completely enclosing the core (2), characterized in that the shell (3) is mixed with an active ingredient (4).

Description

[0001] NANOPILITIES FOR THE ADMINISTRATION OF AN ACTIVE INGREDIENT

[0002] The present invention relates to nanoparticles for administering an active ingredient and a method for producing nanoparticles for administering an active ingredient. The invention further relates to nanoparticles for use as a pharmaceutical and to a pharmaceutical comprising a nanoparticle.

[0003] BACKGROUND OF THE INVENTION

[0004] Drug therapy, along with surgery and radiation therapy, constitutes the most important approach in tumor treatment. For a long time, the systemic administration of cell growth inhibitors was the only option in drug-based tumor therapy. These drugs are distributed throughout the body via the bloodstream through intravenous administration. Instead of this conventional systemic treatment method, drugs can also be targeted directly at tumor cells.

[0005] Magnetizable nanoparticles are now also being used as drug delivery systems for the targeted administration of active substances. For example, US 9,393,396 B2 discloses a method and a composition of coated nanoparticles for the local hyperthermic treatment of tumor cells. The nanoparticles, enriched with an active substance, are guided to a defined anatomical location in the body using a magnetic field, where the nanoparticles can release the active substance.

[0006] Wan et al. describe superparamagnetic iron oxide nanoparticles produced by a polyol process and coated with sodium tripolyphosphate (TPP) in “Stable and Biocompatible Colloidal Dispersions of Superparamagnetic Iron Oxide Nanoparticles with Minium Aggregation for Biomedical Applications”, J Phys Chem C 120 (2016) 23799-23806, https: / / doi.org / 10.1021 / acs.jpcc.6b06614. TPP can be anchored to the oxide surface by forming stable bonds with the phosphate groups. These nanoparticles retain their size, morphology, and non-aggregating properties and can remain stable in aqueous solution for years. These nanoparticles are used as contrast agents.

[0007] Le-Vinh et al. demonstrate in “Alkaline Phosphatase: A Reliable Endogenous Partner for Drug Delivery and Diagnostics” Adv Therap, 5(2) (2022) 2100219, https: / / doi.org / 10.1002 / adtp.202100219 that alkaline phosphatase (ALP) is important for targeted drug delivery and diagnostics. ALP is described as crucial for the activation of phosphorylated pro-drugs, phosphate-directed drug delivery systems, and ALP-triggered probes for diagnostics. The following systems are listed as ALP-related drug delivery systems: surface charge conversion systems, self-assembling nanofibers and hydrogels, reversible sol-gels, multi-reactive hydrogels, and (dis)aggregating nanocarriers.It is also described that polyphosphates can be used in biomedicine for drug delivery and as coating materials, with tripolyphosphate (TPP) and phytic acid (PA) also being mentioned as substrates for ALPs (alkaline phosphatases).

[0008] Du et al. describe magnetic FesCU nanoparticles coated with a biofunctional poly-D-tyrosine phosphate layer in “Enzymatic Transformation of Phosphate Decorated Magnetic Nanoparticles for Selectively Sorting and Inhibiting Cancer Cells”, Bioconjugate Chem. 25(12) (2014), 2129-2133, https: / / doi.org / 10102I / bc500516g. Upon contact with phosphatases, phosphate groups are cleaved off.

[0009] WO 2012 / 057401 Al describes nanoparticles with a magnetizable core and an adenosine triphosphate coating. Gluconic acid can be bound to the nanoparticles.

[0010] DE 43 09 333 Al discloses iron oxide nanoparticles with a polyphosphate and an active ingredient. An extensive list of active ingredients is provided. The examiner also considers this document to be novelty-destroying for numerous claims (1 to 3, 7, 9, 10, 13 and 14).

[0011] WO 2011 / 082796 A2 discloses nanoparticles with different structures with coatings of different polymers (including a polyphosphate) and active substances.

[0012] BRIEF DESCRIPTION OF THE INVENTION

[0013] The treatment methods used in the prior art for the precise delivery of an active ingredient are still insufficient with regard to efficiency, (off-target) cytotoxicity, and efficacy. This is due, among other things, to the fact that the active ingredient cannot distinguish between tumor cells and healthy cells, and the nanoparticles cannot be positioned precisely enough in the body. The active ingredient is neither targeted to the target cells with sufficient specificity, nor can it be (sufficiently) metabolized by delivery to and uptake by the target cells. The object of the present invention is therefore to provide nanoparticles for the administration of an active ingredient in the body that can be positioned more precisely and deliver the active ingredient more precisely to the desired target cells, in order to protect healthy cells and reduce the efficacy and (off-target) cytotoxicity of free active ingredients.

[0014] This problem is solved by nanoparticles for administering an active ingredient, comprising a magnetizable core, wherein the core has a coating made of a polyphosphate, wherein the coating preferably completely encloses the core, characterized in that the coating is impregnated with an active ingredient.

[0015] The term magnetizable core refers both to a core that is suitable for being magnetized and to a permanently magnetic core.

[0016] The term active ingredient refers to both a pure substance and a mixture of active ingredients.

[0017] The inventors discovered that coating the nucleus with a polyphosphate enables the uptake and binding of an active ingredient. The polyphosphate coating allows for the absorption of a defined amount of the active ingredient. Using an external magnetic field, the nanoparticles can be precisely positioned within the body. The polyphosphate coating also allows the release of the active ingredient at the desired location in the body based on physiological conditions.

[0018] The polyphosphate coating is also advantageous because polyphosphate is a biodegradable polymer that is well-tolerated by the body and classified as "Generally Recognized As Safe (GRAS)" by the FDA (US Food and Drug Administration). Polyphosphate is used, among other things, as a dietary supplement.

[0019] For improved biocompatibility, complete encapsulation of the nucleus with the polyphosphate coating is advantageous. The active ingredient is preferentially ionically bound to the polyphosphate. Since polyphosphates are negatively charged or exist as anions at physiological pH in aqueous environments, it is beneficial for the active ingredient to be positively charged or exist as a cation, enabling the active ingredient and the polyphosphate to form an ionic bond. The amount of active ingredient can be stoichiometrically adjusted based on the negative charge carriers of the polyphosphate and the positive charge carriers of the active ingredient. This allows for efficient and controlled uptake of the active ingredient by the polyphosphate. Furthermore, it reduces (off-target) cytotoxicity. The law of mass action results in the polyphosphate-bound active ingredient being released into the physiological environment (body fluid).Because the active ingredient is constantly transported away from the body, it is released continuously. The nanoparticles therefore enable a modified or sustained release of the active ingredient.

[0020] The amount of active ingredient that can be absorbed by the coating can be regulated by the amount of polyphosphate present. This can be achieved, for example, as described above, through the negative charge carriers on the polyphosphate and the positive charge carriers on the active ingredient. In this way, the dosage and dosage accuracy of the active ingredient can be improved while simultaneously reducing cytotoxicity.

[0021] According to the invention, active substances with at least one cationic charge are therefore preferred. This allows the cationically charged active substance to be ionically bound by the anions—namely phosphates—formed by the coating. The cationically charged active substance is preferably selected from the group consisting of cytostatic agents, hormones, antibodies, fusion proteins, peptides, cytokines, signal transduction inhibitors, protease inhibitors, proteasome inhibitors, HMG-CoA reductase inhibitors, analgesics, and mixtures thereof. The aforementioned active substances are advantageous because they cover a broad spectrum of target molecules in the body. For example, a signal transduction inhibitor can be an angiogenesis inhibitor, which promotes the regression of pathological stromal blood vessels in the tumor.

[0022] The system's application is not limited to human treatment but is also possible in veterinary medicine. In one embodiment, the active ingredient is present as a physiologically acceptable salt, and the active ingredient is cationic.

[0023] The core is preferably superparamagnetic or ferritic. Superparamagnetic cores have the additional advantage over ferritic cores that they do not form agglomerated aggregates and remain isolated, making them preferable. In international literature, these are also collectively referred to as SPIONs (superparamagnetic iron oxide nanoparticles).

[0024] In one embodiment, the core comprises, or preferably consists of, Fe2Os or FesCU. In another embodiment, the core comprises, or consists of, maghemite (γ-Fe2O3), cobalt ferrite (CoFe2O4), or mixtures thereof.

[0025] The polyphosphate coating preferably comprises tripolyphosphate (TPP), sodium polyphosphate (PP), pentapolyphosphate, hexametaphosphate, Graham's salt, phytic acid (PA), guanosine triphosphate (GTP), adenosine triphosphate (ATP), and / or mixtures thereof. These polyphosphates are suitable for forming stable bonds with the oxide components of the core. Therefore, these polyphosphates are particularly advantageous when the core comprises, or preferably consists of, Fe₂O₃ or Fe⁻Cl₄. This also renders the core inaccessible to body fluids. The exact bonding mechanism between the oxide component of the core and the polyphosphate is unclear, but it is likely to involve a combination of ionic and hydrogen bonds, possibly also covalent bonds, such that the bond strength is similar to or even stronger than the bond between the negative charge of the phosphate and the positive charge of an active ingredient.This allows the polyphosphate to adhere to the nucleus, even after the active ingredient has been released.

[0026] In a further embodiment, the polyphosphate coating can additionally comprise a compound from the group consisting of dextran, carboxydextran, carboxymethyldextran, PEG-starch, siloxanes, sulfonated styrenedivinylbenzenes, copolymers, and mixtures thereof. The embodiments mentioned in the two preceding paragraphs are particularly advantageous because the nanoparticles retain their size, morphology, and non-aggregating nature. Furthermore, they exhibit long-term storage stability in aqueous solution as well as in lyophilized form.

[0027] The interaction of the components nucleus / coating / active ingredient determines how long the active ingredient can be retained in or on the nanoparticle. In one embodiment, the combination of these three components can be chosen such that, for example, the positively charged active ingredient binds to the negatively charged phosphate group. Simultaneously, the binding of the active ingredient to the phosphate group can be such that, under physiological conditions, the active ingredient can be released into the body and metabolized by the target cells, and that the active ingredient does not remain on the nanoparticle. While the active ingredient is being released, the polyphosphate can remain bound to the nucleus. A balanced binding strength between the active ingredient and the coating can therefore be advantageous. Although polyphosphates bind very strongly to iron oxides [Wan et al., Molecular Mechanism of Linear Polyphosphate Adsorption on Iron and Aluminum Oxide. J. Phys. Chem.C 2020, 124, 52, 28448-28457], it was first shown within the scope of this invention that these polyphosphates can nevertheless be enzymatically cleaved by ALP.

[0028] It is also possible to tailor the mass of the coating to the amount of active ingredient to be administered and the active ingredient itself, so that in one embodiment the layer thickness should be no less than 5%, preferably at least 20%, based on the total mass of the nanoparticle.

[0029] For example, the coated Kem can have a diameter of 50 nm to 850 nm, preferably 100 nm to 800 nm, and particularly preferably 150 nm to 750 nm. This particle size has proven advantageous for the human body, while smaller sizes, such as those in the range of 10 to 30 nm, can also be used in veterinary medicine.

[0030] For applications in human medicine, a core diameter with coating of at least 50 nm is advantageous, as particles with a diameter below 50 nm are often excreted by the human body with considerable delay. Particles smaller than 10 nm have been shown to be capable of penetrating cells and could therefore enter them, which is something the invention aims to prevent.

[0031] The invention also relates to nanoparticles of the aforementioned type for use as pharmaceuticals, for example in human or veterinary medicine, particularly in oncology, immunology, hematology, and / or pain therapy. In oncological and immunological applications, the aforementioned disease patterns are preferred.

[0032] A preferred application in oncology is the treatment of solid tumors. These include, for example, breast cancer, lung cancer, colon cancer, rectal cancer, bladder cancer, hepatocellular carcinoma, ovarian cancer, prostate cancer, melanoma, sarcoma (osteosarcoma, lipsarcoma, leiomyosarcoma), lymphoma, neuroendocrine tumor, germ cell tumor, glioma (astrocytoma, glioblastoma, oligodendroglioma, ependymoma), blastoma, etc. Even though solid tumors are usually localized in one organ, the treatment of their metastases in distant parts of the body is also possible. Furthermore, it is possible not only to treat malignant tumors but also benign tumors, such as myomas, lipomas, adenomas, fibromas, angiomas, chondromas, fibrous dysplasias, giant cell tumors, etc. Therefore, when using nanoparticles as a drug, the active ingredient is preferably a cytostatic agent.

[0033] In another aspect of the invention, the nanoparticles containing the active ingredient are in dried form. Therefore, the invention can also include a kit comprising the aforementioned nanoparticles in one container, a physiologically acceptable buffer in a separate container, and a syringe. The kit is used to prepare a ready-to-use suspension of nanoparticles in the buffer immediately before injection. The physiologically acceptable buffer preferably has a pH value in the range of approximately 3.0 to approximately 10.0, more preferably from approximately 3.5 to approximately 8.0. High storage stability is observed within this range at various storage temperatures. In one embodiment, the pH value of the buffer corresponds to the physiological tissue and blood pH of 7.35 to 7.55.

[0034] In another embodiment, the buffer is a phosphate buffer, a Tris buffer, a citrate buffer, an acetate buffer, preferably a sodium acetate buffer, and is present, for example, at a concentration between approximately 10 mM and approximately 1000 mM. In one embodiment, the nanoparticles and / or the active ingredient of the kit are in lyophilized form. To prepare a ready-to-use infusion solution, the lyophilisate can be reconstituted or diluted by adding a required amount of a buffer, such as 0.9% NaCl solution or 5% glucose solution. In one embodiment, this can be done directly in a vial as a container, i.e., in a vial. This can be advantageous because the lyophilisate is easier to store, has a longer shelf life, and exhibits high storage stability.

[0035] The embodiments mentioned above are not mutually exclusive. For example, in another preferred embodiment, the nanoparticles can be present in a buffer with a physiologically acceptable pH value and the active ingredient can be available as a lyophilisate, or vice versa.

[0036] The invention also relates to a pre-filled syringe comprising

[0037] • a buffer with a physiologically acceptable pH value,

[0038] • Nanoparticles of the type mentioned above containing an active ingredient.

[0039] In one embodiment, the invention provides packaging, such as a cardboard box, containing a pre-filled syringe according to the invention in a blister pack, a needle and optionally instructions for use.

[0040] The invention also relates to a syringe comprising

[0041] • a buffer with a physiologically acceptable pH value,

[0042] • Nanoparticles of the type mentioned above,

[0043] • an active ingredient of the type mentioned above, wherein the active ingredient is positively charged in the buffer with a physiologically acceptable pH.

[0044] In another embodiment, the syringe already contains a nanoparticle with a polyphosphate coating.

[0045] In one embodiment, the inventive device is preferably applied locally. The nanoparticles can either be introduced into the target organ or target tissue via a catheter or injected intradermally, intracutaneously, subcutaneously, intramuscularly, intravitrealally, intralymphatically, intra-articularly, intralumbarly, intraperitoneally, or ventrogluteally using a syringe with a suitable cannula. A trocar can optionally be used.

[0046] In another embodiment, the nanoparticles according to the invention can be administered systemically, for example, intravenously. Alternatively, intravenous administration via a port system is possible. The port system is particularly preferred for oncology patients with poor venous access who require repeated treatment over several cycles. In a further embodiment, the invention can also be administered via a central venous catheter (CVC). A catheter in a larger, central vein can be particularly advantageous for long-term applications or the administration of vasotoxic hyperosmolar substances and cytostatic agents.

[0047] One aspect of the invention also provides a method for positioning nanoparticles of the above-mentioned type, in which a magnetic object, preferably a rod-shaped magnetic object, is positioned on the outside of a body at the epidermis.

[0048] One aspect of the invention also provides a method for positioning nanoparticles of the type mentioned above, which temporarily positions a magnetic object, preferably a rod-shaped magnetic object, on the outer surface of body areas to be treated or on the interface between healthy and body areas to be treated (as part of a surgical procedure).

[0049] The invention also relates to a method for producing a nanoparticle for administering an active ingredient of the type mentioned above. The method comprises the following steps:

[0050] (a) Providing a magnetizable core comprising an iron oxide,

[0051] (b) Providing a mixture of polyphosphate with an active ingredient and

[0052] (c) Coating the magnetizable core with the mixture of polyphosphate and active ingredient.

[0053] Preferably, the nanoparticles obtained after step (c) are dried in a step (d).

[0054] The cores used are particles that are magnetizable or magnetic and can be structured as defined above. The desired thickness of the core and cladding is determined by the cladding mass, which is a proportion of the total mass of the nanoparticle. The total mass of the nanoparticle includes the core and the cladding. The cladding is preferably chosen to comprise at least 5%, and preferably 20%, of the total mass of the nanoparticle.

[0055] The active ingredient containing polyphosphate is preferably applied as an aqueous solution. It is possible for the active ingredient to be applied together with the coating, or it can be subsequently introduced into the coating (e.g., by spraying a dissolved or suspended active ingredient and then drying the solvent or suspension agent).

[0056] Accordingly, the present invention relates to a nanoparticle as defined above or a medicament comprising the nanoparticle for (use in the) treatment of a disease as defined above, the use of the nanoparticle or the medicament in the treatment of a disease as defined above and / or the use of the nanoparticle in the manufacture of a medicament for the treatment of a disease as defined above.

[0057] In another aspect, the present invention relates to a method for treating a disease as defined above, comprising administering a nanoparticle as defined above to a subject who requires it.

[0058] DETAILED DESCRIPTION OF THE INVENTION

[0059] The invention relates to nanoparticles for specific use in therapy, in particular in minimally invasive, localized, personalized and targeted tumor therapy or treatment using suitable active substances, while simultaneously providing maximum protection of healthy cells or adjacent healthy tissue areas, as well as minimal (off-target) cytotoxicity and minimal side effects.

[0060] Further details and advantages of the invention are set out below and with reference to the accompanying figures and the following figure description.

[0061] Fig. 1 shows a schematic representation of the synthesis of the invented nanoparticles. Fig. 2 shows a diagram for the characterization of coated nanoparticles. Fig. 3A shows a diagram of the enzymatic phosphate removal from coated nanoparticles.

[0062] Fig. 3B shows a graph of cell viability.

[0063] Fig. 3C shows a graph of the encapsulation efficiency of ethacridine lactate (ETH) loaded coated nanoparticles.

[0064] Fig. 3D shows a graphic for the characterization of ethacridine lactate (ETH) loaded coated nanoparticles with respect to particle size, polydispersity index (PDI) and zeta potential.

[0065] Fig. 4A shows a schematic representation of the methods used for

[0066] Drug release via dialysis membrane.

[0067] Fig. 4B shows a schematic representation of the method used for

[0068] Active ingredient release via centrifugation.

[0069] Fig. 5 shows a graph of the drug release from drug-loaded nanoparticles.

[0070] Fig. 6A shows a schematic representation of a system according to the invention.

[0071] Fig. 6B shows the schematic application of the system during treatment in the body.

[0072] The treatment method in question is essentially based on two components:

[0073] The first component (Fig. 6A) consists of biocompatible nanoparticles 1 with a magnetizable nucleus 2, in particular with a superparamagnetic or ferritic nucleus, for example Fe2Os or FesCU or maghemite (y-Fe2O3) or cobalt ferrite (CoFe2O4), which are coated with a polyphosphate 3. The polyphosphate coating 3 can be composed of a linear, branched and / or cyclic polyphosphate as well as polyphosphate derivatives.

[0074] The polyphosphate coating imparts an anionic character, enabling the ionic binding of a cationic active ingredient. Particularly suitable polyphosphates include tripolyphosphate (TPP), sodium polyphosphate (PP), pentapolyphosphate, hexametaphosphate, Graham's salt, phytic acid (PA), guanosine triphosphate (GTP), adenosine triphosphate (ATP), and / or mixtures thereof.

[0075] The polyphosphate coating is impregnated with at least one active ingredient. The active ingredient, preferably cationic, is bound to the (anionic) polyphosphate. Polyphosphates can also exist as free acids and therefore lack counterions that would make them salts. For the invention, both salts and free acids can be advantageous in one embodiment; however, salts are preferred because they release the active ingredient in a more controlled manner.

[0076] The second component (Fig. 6B) comprises a magnet 14, for example represented by a magnetizable or magnetic object, preferably a rod-shaped permanent or electromagnet. The magnetic field of the magnet allows the nanoparticles to be guided to the desired location of a body 12 to be treated, in order to direct the nanoparticles containing the active ingredient to target cells, where targeted drug delivery takes place.

[0077] The characteristic dimensions of this component depend primarily on the (spatial) extent or distribution of the target cells or body region to be treated. The geometry of the second component depends on the chosen application technique.

[0078] While the nanoparticles are injected locally or systemically, the magnet is positioned, for example, by inserting it via a puncture instrument such as a trocar near the desired location where the nanoparticle is to be placed. Alternatively, the nanoparticles can be positioned using a magnetizable or magnetic object in the form of a rod that possesses a magnetic field, with the rod being positioned on the outer surface of the body, on the epidermis, at the desired location.

[0079] The invention is particularly suitable for a novel tumor therapy and preferably involves the simultaneous and highly precise application of the two components in the immediate vicinity of the tissue to be treated (nanoparticles are placed intracorporeally, and the magnet is placed intracorporeally or on the body surface, i.e., extracorporeally) from two different positions. The time interval between the application of the two components can also be a defined duration in order to optimize the release of the active ingredient or its effect on the target cells or the body region.

[0080] The drug-loaded nanoparticles are introduced directly into the tissue to be treated, e.g., a tumor, and / or into the immediate vicinity of the tissue to be treated, e.g., the tumor, via the injection methods mentioned above. They then release the drug in a controlled dose to the target cells or target region within the tissue over a defined period. The magnet is positioned near the area of ​​the body to be treated, for example, using a (magnetic) biopsy needle, catheter, and / or trocar. The nanoparticles remain in the body or in the immediate vicinity of the tissue to be treated for a defined period (at least several seconds) and can be removed again using minimally invasive techniques.

[0081] Due to the magnetic effect between the magnet and the nanoparticles, the nanoparticles remain in close proximity to the target cells or organ, where they can achieve maximum efficacy. Drug delivery is enhanced by the movement of the nanoparticles; this movement can be induced externally via the biopsy needle, trocar, catheter, or the magnet on the body surface, for example, by means of oscillation.

[0082] The nanoparticles do not distribute themselves evenly over a wide area of ​​the body, thus ensuring maximum protection of healthy body regions. Due to the highly localized effect of this tumor treatment, it can be simplified as "tumor therapy without off-target cytotoxicity and virtually free of side effects."

[0083] After the treatment period, the nanoparticles can be collected using a magnetic device (e.g., a catheter with a magnetic tip) and gently removed from the body completely or almost completely. The encapsulated nuclei that are not removed with a magnetic device are automatically excreted after a certain period. Because nanoparticles are easily metabolized and extracted from the body, excretion can occur, for example, via the kidneys or via the bile into the intestines and then in the stool. Nanoparticles circulating in the blood can be taken up by the mononuclear phagocyte system via phagocytosis, macropinocytosis, opsonization, and / or caveolin-mediated endocytosis and excreted via various organs such as the liver and / or spleen.

[0084] Synthesis and characterization of nanoparticles (uncoated and unloaded)

[0085] Superparamagnetic iron oxide nanoparticles were prepared by co-precipitation (see Fig. 1). In an exemplary experiment, a 1:2 molar ratio solution of iron(II) and iron(III) ions was first prepared by dissolving 8 mmol of FeCh EEO and 16 mmol of FeCh'öEEO in 150 mL of demineralized water under nitrogen conditions. Iron oxide was then precipitated by the dropwise addition of 15 mL of NFLOH solution (10% w / w) with continuous shaking at 650 rpm until the pH reached a range between 9 and 10 and the solution turned dark black. The mixture was stirred for 1 hour at room temperature. The precipitate was then isolated from the solution using a magnetic rod and washed at least three times with deionized water until a pH of 7 was reached. The purified nanoparticles were then resuspended in 150 mL of demineralized water and stored in the refrigerator for further use.

[0086] To verify the stability of the uncoated nanoparticles, a characterization was carried out with respect to particle size, polydispersity index (PDI) and zeta potential (see Table 1).

[0087] Table 1. Characterization of uncoated nanoparticles (n=3 ±SD).

[0088] The uncoated nanoparticles tended to aggregate in aqueous environments, resulting in particle sizes in the millimeter range. Coating the nanoparticles reduced the particle size by at least a factor of 10, thus reducing it to the nanometer range.

[0089] Synthesis and characterization of coated nanoparticles

[0090] Iron oxide nanoparticles produced by co-precipitation were coated with TPP (tripolyphosphate), PP (sodium polyphosphate), PA (phytic acid), or ATP (adenosine triphosphate). The coating agents were dissolved in demineralized water at various concentrations from 15 mg / mL to 0.25 mg / mL. Uncoated nanoparticles were then added to each coating agent solution in a volume ratio of 1:10 (nanoparticles:polyphosphate). The resulting mixtures were incubated overnight at room temperature with shaking at 600 rpm. The following day, the coated nanoparticles were visually inspected for colloidal stability, separated using a magnet, and washed twice with demineralized water.As part of exemplary orientation studies, small batches of 1 mL coated nanoparticles were produced, while the process was scaled up to 50 mL for the most promising supports for further studies. In this process, 5 mL of uncoated nanoparticles were added to 45 mL of aqueous solutions containing 8 mg / mL TPP, 4 mg / mL PP, 15 mg / mL PA, and 1 mg / mL ATP. The resulting mixtures were treated and purified as described above.

[0091] To verify the stability of the coated nanoparticles, characterization was performed with respect to particle size, polydispersity index (PDI), and zeta potential (see Fig. 2). Nanoparticles coated with TPP, PP, and PA were diluted 1:1 (v / v) with 20 mM HEPES buffer pH 7.4 and preheated to 37°C. Aliquots were taken immediately after preparation and again after a 4-hour incubation at 37°C while shaking at 300 rpm. Following further dilution in water at ratios of 1:10 and 1:5 (v / v) for TPP / PP- and PA-coated particles, respectively, the aliquots were analyzed. ATP-coated nanoparticles were analyzed directly after dilution in a ratio of 1:10 (v / v) with 20 mM HEPES buffer pH 7.4 immediately after preparation and after 4 hours of incubation (37°C and 300 rpm) (n=4 ± SD).

[0092] As shown in Fig. 2, the magnetic nanoparticles were coated with various tested amounts of TPP, PP, PA, or ATP. This prevented particle aggregation, among other things. On the one hand, nanoparticles coated with linear polyphosphate after the addition of coating agents (i.e., TPP, PP, PA, or ATP) showed a lower tendency to aggregate at all concentrations tested, which is underscored by the smaller particle size of approximately 200 nm to 300 nm and the negative zeta potential of approximately -20 mV (see Fig. 2). However, TPP-coated nanoparticles prepared with a coating agent amount of less than 1 mg / mL showed instability after 4 hours of incubation, as evidenced by the zeta potential analysis.Furthermore, aggregation was observed in samples coated with TPP at concentrations below 4 mg / mL after one week of storage at 2°C to 8°C. Therefore, TPP at a concentration of 8 mg / mL was selected for further investigations to obtain more stable coated nanoparticles. For the PP-coated nanoparticles, a coating agent concentration of 4 mg / mL, which exhibits sufficient stability, was selected for further investigation. A PA concentration of 15 mg / mL was chosen for further investigations because it resulted in a smaller particle size and higher stability of the colloidal system compared to the PA-coated nanoparticles at 10 mg / mL. Additionally, the ATP nanoparticles showed sufficient stability when prepared with ATP concentrations below 1 mg / mL, while precipitation was visually observed at higher concentrations.After purification, all samples examined exhibited a particle size of 200 nm to 350 nm, which remained stable for 4 hours. To avoid potential instability, ATP-coated nanoparticles with a concentration of 1 mg / mL were selected for the production of larger batches. All coated nanoparticles exhibited a PDI of less than 0.300, which remained stable over 4 hours of incubation.

[0093] The enzyme-induced phosphate release from coated nanoparticles was determined using the malachite green assay (MLG assay) (see Fig. 3A). Enzyme-induced phosphate release was induced by alkaline phosphatases (ALP), whereby coated nanoparticles were diluted 1:10 (v / v) in 20 mM HEPES buffer and incubated for 48 h with ALP at 2 U / mL and without enzyme. Values ​​reported are mean values ​​of n=4± SD. Significant differences compared to the control samples without ALP are shown as ***p<0.001.

[0094] Figure 3A shows that monophosphate release increased over time during incubation with ALP. In contrast, samples without ALP showed low monophosphate release, confirming the enzyme-induced polyphosphate hydrolysis of the coated nanoparticles. Coating cleavage proceeded more rapidly during the first six hours, followed by a plateau phase, most likely due to enzyme saturation. Overall, after 48 hours, higher monophosphate release was observed for ATP-coated nanoparticles > PA-coated nanoparticles > PP-coated nanoparticles > TPP-coated nanoparticles. Similar values ​​of released monophosphates were found for all coated carriers tested using 2 U / mL and 5 U / mL ALP (alkaline phosphatase). Only PP nanoparticles showed a higher release after 48 hours of incubation with the higher enzyme concentration.A behavior that can also be observed, albeit with a slight tendency, in ATP.

[0095] Cell viability measurements after incubation with coated nanoparticles

[0096] For the purpose of a first cell viability study, the nanoparticles according to the invention were coated with TPP 8:1, PP 4:1, or PA 15:1, as shown in Fig. 3B, the coating not containing any active ingredient for this study. Cell viability was determined using the MTT assay. The MTT assay is a colorimetric test for assessing cell viability by measuring metabolic activity. Viable cells containing NADPH-dependent cellular oxidoreductase enzymes are able to reduce the tetrazolium stain (MTT, yellowish color) to its insoluble formazan (purple color). To measure cell viability in connection with the subject matter of the invention (nanoparticle 1), an immortalized cell line, namely human embryonic kidney cells (HEK 293), was used. The cultivation of HEK 293 cells is known according to the state of the art.In short, HEK 293 cells were seeded in MEM buffer in a 96-well plate and cultured for three days in an atmosphere of 95% relative humidity and 5% CO2. The medium was then removed, the cells were washed with 20 mM HEPES, 268 mM glucose, pH 7.4, and incubated with 100 pL of coated nanoparticles diluted 1:1, 1:10, and 1:50 (v / v) in glucose-HEPES buffer. Additionally, 0.1% Triton X solution and glucose-HEPES buffer were used as positive and negative controls, respectively. The plate was incubated for 24 hours at 37°C (150 rpm). Samples were taken after two, four, and 24 hours, the cells were washed once with glucose-HEPES buffer, and 100 pL of a 0.5 mg / mL MTT solution prepared in glucose-HEPES buffer was added to each well. The plate was incubated for 2.5 hours. The MTT solution was then removed, and the resulting formazan crystals were dissolved in 120 pL of dimethyl sulfoxide (DMSO).100 pL of the resulting solutions were transferred to a 96-well plate, and the absorbance was measured at a wavelength of 540 nm using a microplate reader. Overall, time- and concentration-dependent cell toxicity was observed for almost all nanoparticles tested, as shown in Fig. 3B. Specifically, TPP-coated nanoparticles showed the highest cell viability at dilutions of 1:10 and 1:50 (v / v), followed by PP and PA. At the 1:10 dilution (of the TPP-coated nanoparticles), approximately 90% ± SD of HEK 293 cells were viable after incubation of two and four hours, and approximately 55% ± SD after incubation of 24 hours.

[0097] Production and characterization of drug-loaded coated nanoparticles

[0098] Furthermore, investigations into the fabrication and characterization of drug-loaded coated nanoparticles were carried out (see Fig. 3C). The antiseptic ethacridine lactate (ETH) was selected as a model drug because it has the advantage of being positively charged (i.e., existing as a cation) and is therefore suitable for dislocation or encapsulation onto anionically coated nanoparticles, similar to most cancer drugs. Coated nanoparticles were loaded with different ETH concentrations from 5 pg / mL to 50 pg / mL. A higher encapsulation efficiency was observed for PP and PA, while a lower encapsulation efficiency was seen for TPP-coated nanoparticles and a slightly lower encapsulation efficiency for ATP-coated nanoparticles.In addition, the particle size, polydispersity index (PDI) and zeta potential of the inventive article (active ingredient-loaded coated nanoparticle) were also investigated, with higher stability being observed for samples containing an active ingredient concentration of 20 pg / mL, which were therefore selected for subsequent studies (see Fig. 3D).

[0099] Measurements of drug release

[0100] To assess the influence of enzymatic activity on the drug release of ethacridine lactate (ETH) from loaded nanoparticles, drug release studies were conducted using different methods (see Fig. 4A and Fig. 4B), which are described below.

[0101] First, the drug release profiles were evaluated by physically separating the released drug from the carriers using a dialysis membrane with a specific cut-off that allowed the drug to pass through the membrane into the release medium (see Fig. 4A). Samples were taken from the solution outside the dialysis bag and used to quantify the drug. In the second method, the nanoparticles were separated from the release medium by centrifugation, and the amount of drug released in the supernatant was analyzed (see Fig. 4B). Based on the results of the characterization of the loaded carriers, coated nanoparticles containing 20 pg / mL ETH were obtained. The purified coated ETH-containing nanoparticles collected after centrifugation were resuspended in 1 mL of demineralized water and diluted 1:10 (v / v) with 20 mM HEPES buffer prior to the release studies.

[0102] Figure 5 shows initial investigations into the drug release of drug-loaded nanoparticles across a dialysis membrane. The coating, made of TPP, PP, or PA, was impregnated with the drug. 20 pg / mL ethacridine lactate (ETH) was used after a 1:10 (v / v) dilution in 20 mM HEPES buffer. The ethacridine lactate release (%) was measured after a 24-hour incubation at 37°C with shaking at 300 rpm. The release experiments were performed using a dialysis tube made of regenerated cellulose (14 kDa). The values ​​shown are mean values ​​from n=3 ± SD. Drug release of between 18 and 33% ± SD was demonstrated after 24 hours.

[0103] Another series of experiments, in which release was achieved by centrifugation, yielded a maximum ethacridine lactate (ETH) release of up to 60% ± SD after 48 hours (data not shown). It should be noted that this was an in vitro situation and no external magnetic field was used. It should also be pointed out that the choice of drug for the series of experiments presented here does not preclude the previously mentioned preferred drug classes, and the concentration of 20 pg / mL may also be advantageous for these preferred drug classes.

[0104] Fig. 6A shows a schematic representation of a nanoparticle 1 according to the invention. The nanoparticle 1 for administering a drug comprises a magnetizable or magnetic nucleus 2, which is either superparamagnetic (in the examples studied, it consists of Fe₂O₃ or Fe₃Cr₆ or maghemite (γ-Fe₂O₃)) or ferritic (in the examples studied, it consists of cobalt ferrite (CoFe₂O₄)). The superparamagnetic or ferritic nucleus 2 is completely surrounded by a polyphosphate coating 3. The polyphosphate coating 3 contains at least one active ingredient 4, which is ionically bound to the polyphosphate. The cationic agents studied included cytostatics, hormones, antibodies, fusion proteins, peptides, cytokines, signal transduction inhibitors, protease inhibitors, HMG-CoA reductase inhibitors, analgesics and mixtures thereof.

[0105] In oncological studies, cytostatic agents were used as the active ingredient, which – as shown in Fig. 6B – were injected locally or systemically into the body 10 via a syringe 11, a trocar, a biopsy needle, or a catheter. Locally or systemically within the body 10, the individual nanoparticles 1 are distributed and, in the vicinity of a tumor 12, are guided and held there by means of a magnetic rock of a magnetic source 14, where they can deliver the active ingredient directly to the tumor 12 and exert their cytostatic effect.

Claims

REQUIREMENTS 1. Nanoparticles (1) for administering at least one active ingredient (4), comprising a magnetizable core (2), wherein the core (2) comprises an iron oxide and has a coating (3) made of a polyphosphate, wherein the coating (3) preferably completely encloses the core (2), wherein the coating (3) is provided with a cationic active ingredient (4), wherein the active ingredient (4) is ionically bound to the polyphosphate.

2. Nanoparticles according to claim 1, characterized in that the bond strength between the nucleus and the polyphosphate is stronger than the bond strength between the negative charge of the polyphosphate and the active ingredient.

3. Nanoparticles according to claims 1 to 2, characterized in that the core (2) is superparamagnetic or ferritic.

4. Nanoparticles according to any one of claims 1 to 3, characterized in that the coating (3) comprises polyphosphate tripolyphosphate (TPP), sodium polyphosphate (PP), pentapolyphosphate, hexametaphosphate, phytic acid (PA), guanosine triphosphate (GTP), adenosine triphosphate (ATP) and / or mixtures thereof.

5. Nanoparticles according to one of claims 1 to 4, characterized in that the coating (3) comprises, in addition to the polyphosphate, at least one compound from the group consisting of dextran, carboxydextran, carboxymethyldextran, PEG-starch, siloxanes, sulfonated styrenedivinylbenzenes, copolymer and mixtures thereof.

6. Nanoparticle according to one of claims 1 to 5, characterized in that the nanoparticle has a total mass (Gl) wherein the total mass (Gl) comprises the nucleus (2) and the cladding (3), wherein the total mass of the cladding (3) is at least 5%, preferably at least 20%, based on the total mass (Gl).

7. Nanoparticles according to any one of claims 1 to 6, characterized in that the active substance (4) is selected from the group consisting of cytostatic agent, hormone, antibody, fusion protein, peptides, cytokine, signal transduction inhibitor, protease inhibitor, proteasome inhibitor, HMG-CoA reductase inhibitor, analgesic and mixtures thereof.

8. Nanoparticles according to any one of claims 1 to 7, characterized in that they are in dried form.

9. Nanoparticles according to any one of claims 1 to 8 for use as a pharmaceutical.

10. Nanoparticles for use according to claim 9 for use in human medicine or veterinary medicine, in particular in oncology, immunology, hematology and / or pain therapy.

11. Nanoparticles for use according to claim 9 or claim 10 for a modified release of the active ingredient.

12. Nanoparticles for use according to claim 11, wherein the modified release takes place over a period of 5 minutes to 8 hours.

13. Drug comprising a nanoparticle (1) according to any one of claims 1 to 8 and an active ingredient (4).

14. Method for producing a nanoparticle (1) according to any one of claims 1 to 12, characterized in that, (a) that a magnetizable core (2) is provided, wherein the core (2) comprises an iron oxide, (b) that a mixture of polyphosphate with an active ingredient (4) is provided, and (c) that the magnetizable core (2) is coated with the mixture of polyphosphate with active ingredient (4), preferably the nanoparticles obtained after step (c) being dried in a step (d).

15. Kit, comprehensive • Nanoparticles according to any one of claims 1 to 12 in a container, • a physiologically acceptable buffer in a separate container, as well as • a syringe, preferably for use in the preparation of a ready-to-use suspension from the nanoparticles in the buffer immediately before injection of the suspension.

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